Project Summary/Abstract Lyme disease is a tick-borne infection caused by the spirochete Borrelia burgdorgeri sensu lato (Bbsl) and is the most prevalent vector-borne disease in the United States. The latest surveillance from the Centers for Disease Control reported 40,000 new cases of Lyme disease in 2017 alone. Alarmingly, the disease is expanding from its endemic areas in the Northeast, mid-Atlantic, and Upper Midwest to neighboring states. The rapidly increasing public health risks and rising healthcare costs associated with the disease are exacerbated by controversies surrounding its diagnosis and treatment, especially in cases of `post-treatment Lyme disease syndrome' and `chronic Lyme disease'. In these cases, there are no definitive consensus diagnostic markers that are both sensitive and specific for the infection. While the under-diagnosis of Lyme disease has naturally lead to under-treatment, the mis-diagnosis and mis-treatment of the condition has led to serious morbidity as well. Taken together, the data make it clear that the development of new diagnostic and theranostic tools for Lyme disease is an urgent, unmet clinical need. This R21 proposal is focused on the design, synthesis, and in vitro evaluation of sensitive and specific radiotracers for the diagnostic and theranostic positron emission tomography (PET) of patients with Lyme disease. The target for these radiopharmaceuticals will be VlsE, a protein that is abundantly expressed on the surface of Bbsl throughout its time in its vertebrate host. Specific Aim 1 (SA1) will be focused on the identification of broadly reactive VlsE-based biomarkers via the comparative analysis of BBsl genomes, with an overall goal designing 15 candidate targets (10 variants of VlsE and 5 vls-based peptides) based on consensus and conserved sequences. Specific Aim 2 (SA2) will be centered on the expression of the candidate VlsE proteins, the synthesis of the vls-encoded peptides, and the in vitro testing of the antigenicity of these biomolecules with patient antisera in order to identify the most broadly reactive target molecules. Specific Aim 3 (SA3) will focus on the generation of a VlsE-specific monoclonal antibody and F(ab) fragment as well as the radiosynthesis of the corresponding radioimmunoconjugates labeled with positron-emitting radiometals zirconium-89 (89Zr) and gallium-68 (68Ga). The in vitro immunoreactivity of these radioimmunoconjugates will be interrogated with several strains of VlsE-expressing BBsl as well as control gram-negative and gram-positive bacteria. Ultimately, we believe that this proposal could have a transformational impact on both the basic science and clinical management of Lyme disease. In the laboratory, a Lyme-targeted radiotracer could be a valuable research tool during the development of murine models of the disease and the evaluation of Bbsl-targeted therapeutics. Even more importantly, in the clinic, a VlsE-targeted radiopharmaceutical could prove to be both a useful diagnostic tool to help identify patients with active infections and a valuable theranostic tool to monitor the response of patients to therapy.